Pathogenic microorganisms are rapidly becoming resistant to many of the antibiotics that we have used for decades to treat disease. This is especially true for pathogens that cause hospital-acquired (nosocomial) infections. New antibiotics are needed, but where does one look? One untapped source is microorganisms from extreme environments. The long-term objectives of this proposal are to isolate microorganisms from hypersaline environments and determine if they produce new classes of antimicrobials suitable for fighting disease. The specific aims involved in these objectives include determining if these isolates are new, how closely related to existing organisms they are, if they produce new antibiotics, purifying and characterizing these new antibiotics, determining which bacterial organisms they inhibit, and cloning the genes that produce these new antimicrobials. The achievement of these goals will require the following methods. First, an efficient protocol for recovering hundreds of extreme halophiles from various hypersaline environments (surface salt deposits, inside salt crust and from brine) must be in place. Second, the Polymerase Chain Reaction (PCR) employing domain-specific primer pairs will be used to amplify either the 16S or 18S genes from the newly purified organisms. The DNA sequence of these genes will place the organisms taxonomically with respect to existing isolates. Third, organisms that produce inhibitory products will be found by an antagonism study. This involves placing a small amount of culture onto "lawns" (a plate of growth medium containing a thin layer of organism spread onto the surface) of all of the other extremely halophilic isolates and looking for the presence of zones of inhibition after incubation. In addition, the same test will be done by challenging the new isolates against lawns of various gram positive and gram negative organisms. Fourth, the material producing the zone of inhibition will be purified using a series of biochemical methods including concentration of culture supernatants by filters of smaller and smaller pore sizes, gel filtration column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reversed-phase and ion exchange high performance liquid chromatography (HPLC). Finally, genes that encode these new antibiotics will be cloned by synthesizing degenerate oligonucleotide probes based upon the amino acid sequence of the purified antimicrobial. The probes will be rendered radioactive and used to search for restriction fragments that hybridize to the probe and contain the antibiotic (this process is called Southern blotting). The fragments containing the new genes will be cloned and sequenced and the new genes subjected to genetic and physiologic analysis. At a very rapid rate, disease-causing microorganisms are becoming resistant to the antibiotics currently in use. The relevance of this proposal to public health is tapping a new reservoir of microorganisms that produce new antibiotics. The new reservoir is microorganisms that thrive in very high salt (hypersaline) environments like the Great Salt Lake, UT. [unreadable] [unreadable] [unreadable]